Amplification of ribonucleic acids

ABSTRACT

The invention relates to methods for the amplification of ribonucleic acids, comprising the following steps: (a) a single stranded DNA is produced from an RNA by means of reverse transcription, using a single-stranded primer having a defined sequence, an RNA-dependent DNA polymerase and deoxyribonucleoside triphosphates; (b) the template RNA is removed; (c) a DNA duplex is produced by means of a single-stranded primer comprising a box sequence, a DNA polymerase and deoxyribonucleoside triphosphates; (d) the duplex is separated into single-stranded DNAs; (e) DNA duplexes are produced from one of the single-stranded DNAs obtained in step (d) by means of a single-stranded primer comprising a promoter sequence at its 5′end and the same defined sequence as the primer used in step (a) at its 3′end, a DNA polymerase and deoxyribonucleoside triphosphates; (f) a plurality of RNA single strands, both ends of which comprise defined sequences, are produced by means of an RNA polymerase and ribonucleoside triphosphates. The invention also relates to kits for amplifying ribonucleic acids according to one of said methods, said kits comprising the following components: (a) at least at least one single-stranded primer, which contains a promoter sequence; (b) at least one single-stranded primer comprising a box sequence; (c) an RNA-dependent DNA polymerase; (d) deoxyribonucleoside triphosphates; (e) a DNA-dependent DNA polymerase; (f) an RNA polymerase; and (g) ribonucleoside triphosphates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. § 371of International Application Number PCT/EP03/05579, filed May 27, 2003,the disclosure of which is hereby incorporated by reference in itsentirety, and claims the benefit of German Patent Application Number 10224 200.3, filed May 31, 2002.

INCORPORATION OF SEQUENCE LISTING

A paper copy of the Sequence Listing and a computer readable form of thesequence listing on diskette, containing the filed named“SL19006004.txt”, which is 1,943 bytes in size (measured in MS-DOS), andwhich was recorded on Nov. 29, 2004, are herein incorporated byreference.

BACKGROUND OF THE INVENTION

To date, a multitude of processes resulting in the amplification ofnucleic acids are known. The best known example is the polymerase chainreaction (PCR), developed by Kary Mullis in the mid-eighties (see Saikiet al., Science, Vol. 230 (1985), 1350-1354; and EP 201 184).

During the PCR reaction, single-stranded primers (oligonucleotides witha chain-length of usually 12 to 24 nucleotides) bind to a complementary,single-stranded DNA sequence. These primers are subsequently elongatedto double stranded DNA, in the presence of a DNA polymerase anddeoxyribonucleoside triphosphates (dNTPs, namely dATP, dCTP, dGTP anddTTP). The double stranded DNA is separated by heating into singlestrands. The temperature is reduced sufficiently to allow a new step ofprimer binding. Again, primer elongation results in double stranded DNA.

Repetition of the steps described above enables exponentialamplification of the input DNA. This is achieved by adjusting thereaction conditions such that almost each molecule of single-strandedDNA within each round of amplification will be transformed into doublestranded DNA, melted into single-stranded DNAs which will be used againas template for the next round of amplification.

It is possible to conduct a reverse transcription reaction prior to theabove mentioned PCR reaction. This means, in the presence of anRNA-dependent DNA polymerase mRNA is transformed into single-strandedDNA (cDNA), which can then be used in a PCR reaction, hence resulting inthe amplification of RNA sequences (see EP 201 184).

This basic reaction model of a PCR reaction has been altered in the lastyears and a multitude of alternatives have been developed, depending onthe starting materials (RNA, DNA, single or double stranded) and alsorelating to different reaction products (amplification of specific RNAor DNA sequences from the mixture of different nucleic acids within onesample, or the amplification of all RNA/DNA sequences present in onesample).

Over the last years, so called microarrays for the analysis of nucleicacids are used with increasing frequence. The essential component ofsuch a microarray is an inert carrier onto which a multitude ofdifferent nucleic acid sequences (mostly DNA) were bound in differentregions of the carrier. Usually, within one particular very smallregion, only DNA with one specific sequence is bound, resulting inmicroarrays with several thousand different regions capable of bindingseveral thousand different sequences.

These microarray plates can be incubated with a multitude of nucleicacid sequences (mostly also DNA) obtained from a sample of interest.Resulting, under suitable conditions (ion content, temperature and soforth), in complementary hybrid molecules of nucleic acid sequences fromthose sequences originating form the sample of interest and thosesequences bound to the microarray plate. Unbound, non-complementarysequences can be washed off. The regions on the microarray containingdouble stranded DNA can be detected and thus, the sequences as well asthe amount of nucleic acids bound from the original sample can beanalysed.

Microarrays are used to analyse expression profiles of cells, henceallowing the analysis of all mRNA sequences expressed in certain cells(see Lockhart et al., Nat. Biotechnol. 14 (1996), 1675-1680).

The amount of mRNA available for this sort of analysis is usuallylimited. Therefore special processes have been developed to amplify theribonucleic acids, which will be analysed by means of microarrays. Tothis end, ribonucleic acids will possibly be converted to more stablecDNAs by means of reverse transcription.

Methods, yielding large amounts of amplified RNA populations of singlecells are described in e.g. U.S. Pat. No. 5,514,545. This method uses aprimer containing an oligo-dT-sequence and a T7-promoter region. Theoligo-dT-sequence binds to the 3′-poly-A-sequence of the mRNA initiatingthe reverse transcription of the mRNA. Alkaline conditions result in thedenaturation of the RNA/DNA heteroduplex, and the hairpin structure atthe 3′-end of the cDNA can be used as primer to initiate synthesis ofthe second DNA strand. The resulting construct is converted to a lineardouble stranded DNA by using nuclease S1 to open the hairpin structure.Then the linear double stranded DNA is used as template for T7 RNApolymerase. The resulting RNA can be used again as template for thesynthesis of cDNA. For this reaction oligonucleotide hexamers of randomsequences (random primers) are used. Following heat-induceddenaturation, the second DNA strand is produced by means of the abovementioned T7-olido-dT-primer and the resulting DNA can again be usedagain as template for T7 RNA polymerase.

An alternative strategy is presented in U.S. Pat. No. 5,545,522. Here,it is demonstrated that a single oligonucleotide primer can be used toyield high amplifications. RNA is reverse transcribed to cDNA, and theprimer has the following characteristics: a) 5′-dN₂₀, meaning a randomsequence of 20 nucleotides; b) a minimal T7-promoter; c) GGGCG astranscription-initiation sequence; and d) oligo-dT₁₅. Synthesis of thesecond DNA strand is achieved by partial RNA digestion by RNase H. Theremaining RNA-oligonucleotides are used as primers for DNA polymerase I.The ends of the resulting DNA are blunted by T4-DNA polymerase.

A similar procedure is disclosed in U.S. Pat. No. 5,932,451. In thisprocedure, two so-called box-primers are added within the 5′ proximalarea, enabling the double immobilisation by using biotin-box-primers.

However, the above mentioned methods to amplify ribonucleic acids havemajor disadvantages. All of the above mentioned methods result in RNApopulations which are different from the RNA populations present in theoriginal starting material. This is due to the use of theT7-promoter-oligo-dT-primers, which do primarily amplify RNA sequencesof the 3′-section of the mRNA. Furthermore, it has been shown that thoseextremely long primers (more than 60 nucleotides) are prone to buildprimer-primer-hybrids and they do also allow for non-specificamplification of the primers (Baugh et al., Nucleic Acids Res. 29 (2001)E29). Therefore the known procedures result in the production of amultitude of artefacts, interfering with the further analysis of thenucleic acids.

The problem underlying the present invention therefore resides inproviding a method to amplify ribonucleic acids, which allowshomogeneous and in particular highly reproducible amplification of theribonucleic acids present in the starting material.

This problem is now solved using a method comprising the followingsteps:

-   -   a) a single stranded DNA is produced from an RNA by means of        reverse transcription, using a single-stranded primer having a        defined sequence, an RNA-dependent DNA polymerase and        deoxyribonucleoside triphosphates;    -   b) the template RNA is removed;    -   c) a DNA duplex is produced by means of a single-stranded primer        comprising a box sequence, a DNA polymerase and        deoxyribonucleoside triphosphates;    -   d) the duplex is separated into single-stranded DNAs;    -   e) DNA duplexes are produced from one of the single-stranded        DNAs obtained in step (d) by means of a single-stranded primer        comprising a promoter sequence at its 5′end and the same defined        sequence as the primer used in step (a) at its 3′end, a DNA        polymerase and deoxyribonucleoside triphosphates; and    -   f) a plurality of single stranded RNAs is produced, both ends of        which comprise defined sequences, by means of an RNA polymerase        and ribonucleoside triphosphates.

BRIEF SUMMARY OF THE INVENTION

The present invention includes a process for the amplification ofribonucleic acids, comprising the following steps: (a) a single strandedDNA is produced from an RNA by means of reverse transcription, using asingle-stranded primer having a defined sequence, an RNA-dependent DNApolymerase and deoxyribonucleoside triphosphates; (b) the template RNAis removed; (c) a DNA duplex is produced by means of a single-strandedprimer comprising a box sequence, a DNA polymerase anddeoxyribonucleoside triphosphates; (d) the duplex is separated intosingle-stranded DNAs; (e) DNA duplexes are produced from one of thesingle-stranded DNAs obtained in step (d) by means of a single-strandedprimer comprising a promoter sequence at its 5′end and the same definedsequence as the primer used in step (a) at its 3′end, a DNA polymeraseand deoxyribonucleoside triphosphates; and (f) a plurality of singlestranded RNAs is produced, both ends of which comprise definedsequences, by means of an RNA polymerase and ribonucleosidetriphosphates. The present invention further provides kits foramplifying ribonucleic acids according to one of said processes, saidkits comprising the components required for performing the processes ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth a schematic diagram of the process of the presentinvention.

DESCRIPTION OF THE NUCLEIC ACID SEQUENCES

-   SEQ ID NO: 1 sets forth a nucleic acid sequence of a primer of the    present invention.-   SEQ ID NO: 2 sets forth a nucleic acid sequence of a primer of the    present invention.-   SEQ ID NO: 3 sets forth a nucleic acid sequence of a primer of the    present invention.-   SEQ ID NO: 4 sets forth a nucleic acid sequence of a primer of the    present invention.-   SEQ ID NO: 5 sets forth a nucleic acid sequence of a primer of the    present invention.-   SEQ ID NO: 6 sets forth a nucleic acid sequence of a primer of the    present invention.

DETAILED DESCRIPTION OF THE INVENTION

It was surprisingly found that the above combination of steps leads to aremarkably homogeneous amplification of the ribonucleic acids present inthe starting material. At the same time the process according to theinvention prevents the production of artefacts. Hence the processaccording to the invention is a substantial improvement of methods toamplify nucleic acids and allows at the same time the improvement ofprocedures to analyse ribonucleic acids by means of microarrays.

The process according to the invention results in the amplification ofsingle-stranded ribonucleic acids which have the inverse orientation(antisense sequence) when compared to the ribonucleic acids present inthe starting material.

According to one embodiment of the processes of the invention, theribonucleic acids used as template in step (a) were isolated from cellsof an organism. The isolation of mRNA from cells of multi-cellularorganisms is especially preferred.

The single-stranded primer used in (a) is a primer of any definedsequence, this mans that this primer does not contain a random sequenceof nucleotides. Also more than one defined primer can be used.

The single-stranded primer described in (a) contains preferably anoligo-dT-sequence, a sequence containing several dT-nucleotides. Thishas the advantage that the primer binds to the poly A-tail of eukaryoticmRNA. This results in reverse transcription of almost exclusively mRNA.

In the process according to the invention it is preferred if the primerdescribed in (a) contains a 5′-(dT)₁₈V sequence. This is a primer with18 dT-deoxyribonucleotide-monomers followed by a singledeoxyribonucleotide of different nature (namely dA, dC, or dG, herereferred to as V). This primer nearly exclusively allows reversetranscription of sequences which are located in the close vicinity ofthe 5′-end of the polyA-tail. Different to known processes, the use ofsuch a primer therefore suppresses the production of artefacts resultingfrom further downstream primer binding of normal oligo-dT-primers withinthe large polyA-areas of mRNAs.

Alternatively, the primer of (a) can be homologous to one or severalspecific sequences, present in the sample. In this procedure, theamplification of ribonucleic acids is limited to specific targetsequences.

Further, in the process according to the invention, it is preferred ifthe RNA-part of the DNA-RNA-hybrids described in (b) is digested byRNase. For this procedure any RNase can be used. The use of RNase Iand/or RNase H is preferred. This step results in the elimination of allRNAs which have not been transcribed into cDNA during the first step ofthe procedure, particularly ribosomal RNAs, but also all other cellularRNAs which do not have the polyA-tail, characteristic for mRNAs.

The DNA-RNA-hybrids, which result from the reverse transcriptionreaction can also be separated into single strands by means of heat.However, different to heat treatment, the use of RNases has the furtheradvantage that genomic DNA present in the sample is not converted tosingle-stranded form, and thus it will not act as a hybridisationtemplate for the primers used in the following steps of the procedure.Special advantages result from the use of RNase I, because this enzymecan easily be inactivated at temperatures below those resulting indenaturation of the genomic DNA. The aim of the process according to theinvention is the amplification of ribonucleic acids, hence the use of astable RNase could hinder this process and would necessitate eliminationby elaborate procedures.

In step (c) a single-stranded primer is used, which contains a Boxsequence. Within the scope of the present invention an RNA- orDNA-sequence is called a Box sequence if it comprises a defined sequenceof 10 to 25 nucleotides, having only low homology to gene sequences ofthe organisms from which the starting RNA template for amplification wasisolated from.

Low homology between a potential Box sequence and corresponding genesequences can be determined experimentally by means of standard NorthernBlot analysis. To this end RNA samples from an organism of interest(e.g. plants, humans or animals), this means the organism from which RNAwas isolated for further amplification, is separated by means ofelectrophoresis and transferred onto a membrane and hybridised with alabelled oligonucleotide containing the Box sequence. Low homology ischaracterised by the absence of a hybridisation signal under stringenthybridisation conditions. For example, stringent conditions can beachieved by washing the membrane, after the hybridisation, for 40minutes at 25° C. with a buffer containing 0.1*SSC and 0,1% SDS.

As an alternative to the above mentioned experimental procedure toverify a Box sequence, it is possible to determine a sequence with lowhomology by searching databases containing known gene sequences, thatare expressed in multi-cellular organisms. To date, all known genesequences that are expressed in multi-cellular organisms are stored indatabases with open access to the public. These sequences are eitherstored as gene sequences with known function, or if the function is notknown as so called “expressed sequence tags” or ESTs. A sequence withonly low homology to known sequences is suitable as a Box sequence, ifthis sequence in comparison to all sequences listed in a database, showsover a total length of 10 to 25 nucleotides at least 20%, but preferably30 or 40%, differences in their sequences. This means that over a lengthof 10 nucleotides at least 2 nucleotides are different, and 4 over alength of 20 nucleotides, respectively. Sequence identities, ordifferences between 2 sequences are preferably determined using theBLAST software.

Therefore, a certain sequence can be determined as a Box sequence for acertain use. If human mRNA is to be amplified in the process accordingto the invention, the described low homology has to be determined bycomparison with a human database or hybridising human RNA with the Boxsequence in a Northern Blot. If plant mRNA is to be amplified in theprocess according to the invention, the described low homology has to bedetermined by comparison with plant ribonucleic acids. A sequence,suitable as a Box sequence, in a certain uses of the process accordingto the invention, therefore might not be suitable as Box sequence in adifferent use.

The Box sequence is preferably selected not to contain viral sequences,neither coding nor regulatory sequences (promoter or terminatorsequences) of viruses or bacteriophages.

In the process according to the invention, use of a primer comprising asuitable Box sequence is highly advantageous, because this drasticallyreduces the production of amplification artefacts.

The Box sequence is located in the 5′ region of the primer used in step(c). Preferably the primer further contains a sequence of 3 to 6 randomnucleotides (N-3-N6), and a defined trinucleotide sequence (for exampleTCT). Alternatively, a mix of different trinucleotide sequences can beincluded in the primer.

Preferably, the primer containing the Box sequence has a length of 40nucleotides, a length of 30 nucleotides is especially preferred.

In an especially preferred version of the process according to theinvention, a single-stranded primer is used in step (c) comprising inaddition to the Box sequence an especially suitable sequence of at least6 nucleotides. The primer to be used in step (c) can, for example, havethe following sequence: GCA TCA TAC AAG CTT GGT ACC N₃₋₆ TCT (27-30 nt).

In steps (c) and (e), any DNA-dependent DNA polymerase can be used.Preferably, a reverse transcriptase is used. It is especiallyadvantageous in the process according to the invention, to use a reversetranscriptase, because this DNA polymerase does not separate doublestranded DNA. For the DNA polymerisation in steps (a), (c) and (e) alsodeoxyribonucleoside triphosphates are needed, usually dATP, dCTP, dGTPand dTTP.

In step (d), separation of double stranded DNA into single strands canbe achieved by any procedure. However, this is preferably done by meansof heat.

In step (e) a single-stranded primer is used, which contains a promotersequence. A promoter sequence allows the binding of the RNA polymeraseand initiates the synthesis of an RNA strand. Preferred in (e) is theuse of a single-stranded primer containing the sequence of a highlyspecific RNA polymerase promoter like T7, T3 or SP6. A primer with aT7-promoter has for example the following sequence: ACT AAT ACg ACT CACTAT A g⁺¹ g (dT)₁₈V (40 nt).

Selecting an RNA polymerase to be used in the method of the presentinvention in step (f) depends on the promoter sequence used in theprimer sequence. If the primer contains a T7 polymerase sequence, then aT7 RNA polymerase has to be used in step (f).

To obtain ribonucleic acids in step (f), ribonucleotide-monomers arefurther needed, usually ATP, CTP GTP and UTP.

For the first time, the process according to the invention allows astrong and specific amplification of the starting RNA sequences,representing the total sequences of the entire RNA population. Theamplification factor of the starting RNA sequence is at least 500,whereas a factor of more than 1000 is especially preferred.

The present invention also includes processes according to theinvention, which result in removal of single-stranded primers and primerinduced artefacts (e.g. primer-dimers), before the RNA polymerase isadded.

Further amplification of ribonucleic acids can be achieved in processeswherein the following steps are performed after step (f):

-   (g) single-stranded RNAs generated in step (f) are used as a    template to synthesize single-stranded DNA by means of reverse    transcriptase, a single-stranded primer, containing the Box    sequence, an RNA-dependant DNA polymerase and deoxyribonucleoside    triphosphates;-   (h) the RNA is removed;-   (i) using the in (h) generated single-stranded DNA as template,    double-stranded DNA is synthesised using a single-stranded primer,    comprising a promoter sequence in its 5′ region and the same defined    sequence as the primer used in step (a), in its 3′ region, a DNA    polymerase and deoxyribonucleoside triphosphates;-   (j) a multitude of single-stranded RNAs are synthesized using a RNA    polymerase and ribonucleoside triphosphates.

This variation of the process according to the invention has specificadvantages. The defined sequence at the ends of the ribonucleic acids,produced in steps (a) to (f) in the process facilitates reversetranscription into DNA. This DNA can be used as template for further,promoter-based RNA synthesis. In this manner, a further at least 50-foldincrease of the amount of amplified ribonucleic acids can be achieved.

Preferably the process according to the invention is performed such thatthe single-stranded primer used in step (i) has the same sequence as theprimer used in step (a).

The primer used in step (g) can be identical to the primer used in step(c). Alternatively, the primer used in step (g) can consist only of thewell defined Box sequence, and does not include the less specificelements of the primer used in step (c). The primer used in step (g)can, for example, have the following sequence: GCA TCA TAC AAG CTT GGTACC (21 nt).

The RNA produced in step (h) can be removed with any known, appropriateprocedure, however, hydrolysis with RNase is preferred.

Before proceeding with the transcription reactions (steps (f) and (j)according to the process of the invention) it may be advantageous to useany known procedures for purifying the nucleic acids thus generated.During such a purification procedure, special care should be taken thatany excess of primers and/or primer induced artefacts (e.g. primerdimers) are removed.

The process according to the invention as described above producesexclusively single-stranded RNA with antisense orientation (so calledantisense strands). The present invention also covers process, which bymeans of to date known standard processes (reverse transcription, PCR,cDNA second-strand synthesis, transcription and so forth) convert thesingle-stranded antisense RNA into double-stranded DNA, single-strandedDNA of any orientation, or into single-stranded RNA withsense-orientation (so called sense-strand). The precise manner of theprocedure and the resulting product is highly dependant on the intendeduse.

The present invention also relates to kits comprising all reagentsneeded to amplify ribonucleic acids by means of the process according tothe invention. These kits kits may comprise the following components:

-   -   (a) at least one single-stranded primer, comprising a promoter        sequence;    -   (b) at least one single-stranded primer, comprising a Box        sequence    -   (c) an RNA-dependent DNA polymerase;    -   (d) deoxyribonucleoside triphosphates;    -   (e) a DNA-dependent DNA polymerase;    -   (f) an RNA polymerase; and    -   (g) ribonucleoside triphosphates

Accordingly, the kit contains at least two different single-strandedprimers, which are characterised by the above mentioned criteria.However, dependent on the intended use, the kit may contain more thantwo primers and additional reagents.

In addition, the kit may contain RNase I and/or RNase H.

The kit contains a DNA polymerase, preferably a reverse transcriptase orany other DNA polymerase, which does not separate double-stranded DNA.

The kit may further comprise a composition for DNA-labelling with adetectable moiety and one or more DNA microarrys. The kit may thuscontain all components necessary to perform gene expression analysis. Ingeneral, the different components of the kit will be supplied indifferent tubes. However, components used in the same step of theprocedure may also be supplied in one tube.

Therefore, the present invention further relates to procedures for theanalysis of nucleic acids, during which ribonucleic acids are obtainedand amplified using any of the procedures described in the presentinvention and which will thereafter be analysed using a microarraytechnique. Ribonucleic acids are normally isolated form biologicalsamples. Prior to microarray analysis, ribonucleic acids amplified bytechniques described in the present invention might be transcribed intocDNA, using a reverse transcription. The present invention allowsanalysis of amount and/or sequence of the cDNA. The DNAs obtained in theintermediate steps can also be used, for example, to generate, by meansof cloning, a representative genebank, containing genes derived from abiological sample or genes derived from a sample produced in alaboratory.

FIG. 1 illustrates an example of the procedures of the present inventionas a schematic diagram: In a first step RNA is transcribed intosingle-stranded DNA by means of reverse transcription, using an anchoredoligo(dT)₁₈V primer. This procedure allows the reverse transcriptionstarting at the transition of the ploy-A tail of the mRNA to the 3′-UTRarea. The next step eliminates the RNA from the RNA-cDNA-heteroduplex byuse of RNase H/RNase I and the remaining RNA (mainly ribosomal RNA) isdigested by RNase I.

Synthesis of the second, complementary DNA strand is used to introducethe Box sequence via a specific primer. This primer consists in one partof 6-9 random nucleotides and a second part which comprises the Boxsequence.

After primer annealing, elongation to double stranded DNA is achieved byincubation with DNA polymerase. Excess primers are removed andheat-induced denaturation of the DNA double strand is followed by areduction of the incubation temperature, enabling a primer containingthe T7-promoter and a (dT)₁₈V sequence to hybridise with the DNA. Afurther DNA strand is obtained by primer elongation. Hereafter excessprimer and primer-induced artefacts (primer dimers) are removed and theRNA amplification is achieved by in vitro transcription utilizing the T7promoter.

FIG. 1 c describes the procedure to amplify ribonucleic acids accordingto the above mentioned steps (g-j). The ribonucleic acid produced instep (f) is reverse transcribed, using a primer containing the Boxsequence, a reverse transcriptase and dNTPs. RNases remove theRNA-strand. Using a primer, containing a promoter and the oligo-dTsequence, a second DNA strand is produced, that is used as template forthe RNA polymerase in the transcription reaction.

The order and detailed implementation of the reaction steps of thepresent invention are illustrated by the Examples:

EXAMPLES Example 1 First amplification round (see, e.g., FIGS. 1 a, 1 b)Example 1A Reverse Transcription of 100 ng Total-RNA UsingOligo(dT)₁₈V-primer

First strand-DNA-Synthesis: RNA (50 ng/μl):   2 μl Oligo(dT)₁₈ V(5pmol/μl): 1.5 μl dNTP-Mix (10 mM):   1 μl DEPC-H₂O 3.5 μl

Incubate 4 min at 65° C. in a thermocycler with a heated lid, then placeimmediately on ice. Mastermix for synthesis of the 1^(st) strand of cDNA5 × RT-buffer 4 μl RNase-inhibitor (20 U/μl) 1 μl Superscript II (200U/μl) 1 μl DEPC-H₂O 6 μl

Pipette components for the mastermix on ice and add to the tubecontaining the reverse transcription mix. Place samples in athermocycler (preheated to 42° C.)

Incubate as follows:

-   37° C./5 minutes-   42° C./50 minutes-   45° C./10 minutes-   50° C./10 minutes-   70° C./15 minutes (enzyme inactivation)

Place samples on ice.

Example 1B RNA Removal

Removal of RNA from the reaction First strand-cDNA mix 20 μl RNase-Mix(RNase H/RNase I; each at 5 U/μl)  1 μl

Incubate for 20 min at 37° C., hereafter place samples on ice. RNase Awas not used for RNA elimination, because RNase A is not readilyinactivated. RNase I on the other hand, the enzyme used in thisinvention, can be inactivated easily and completely by incubation at 70°C. for 15 min.

Example 1C Double-Stranded Template DNA with Box-Random-Primer andT7-(dT)₁₈V

Random priming of first strand cDNA with Box-random Primer Firststrand-cDNA 21 μl  dNTP-mix (10 mM) 1 μl Box-random-primer (10 pmol/μl)1 μl 10 × polymerase buffer 6 μl H₂O 20 μl 

Incubation:

-   70° C./1 minute-   37° C./1 minute-   add 1 μl reverse transcriptase (5 U/μl) to each sample-   incubate at 37° C./30 minutes

Removal of excess primer

-   1 μl Exonuclease I (10 U/μl)-   37° C./5 minutes-   96° C./6 minutes

Place samples on ice

Double-stranded template DNA with T7-(dT)₁₈V

-   2 μl T7-(dT)₁₈V primer (10 pmol/μl)-   70° C./1 minute-   42° C./1 minute-   add 1 μl reverse transcriptase (5 U/μl) to each sample-   42° C./30 minutes-   cool to 37° C.-   1 μl T4 DNA polymerase (10 U/μl)-   37° C./1 minute-   65° C./1 minute

Place samples on ice.

Example 1D Purification of the cDNA with High-Pure PCR Purification Kit(Roche)

cDNA purification Reaction mix  50 μl Binding-buffer 250 μl Carrier(cot-1-DNA, 100 ng/μl)  3 μl

Transfer mix onto provided columns, spin in a tabletop centrifuge atmaximal rpm for 1 min. Discard the flow-through. Add 500 μl washingbuffer to the column and spin as above, discard flow-through and repeatthe wash step with 200 μl washing buffer. Transfer columns onto a new1.5 ml reaction tube add 50 μl elution buffer, incubate for 1 min at RTand centrifuge as described above. Repeat the elution step once, againusing 50 μl buffer.

Example 1E Ethanol Precipitation of Purified cDNA

Do not vortex the Pellet Paint™-carrier stock solution and store in thedark. Keep at −20° C. for long term storage, smaller aliquots can bestored for approximately 1 month at 4° C. Ethanol precipitation Eluate100 μl Carrier (Pellet Paint ™)  2 μl Sodium acetate  10 μl Ethanol;absolute 220 μl

Mix thoroughly (do not vortex) and pellet cDNA by centrifugation atmaximal rpm for 10 min at RT. Discard supernatant; wash pellet once with200 μl of 70% ethanol. Centrifuge for 1 min as described above. Removesupernatant completely using a pipette. Dry pellet by incubation of theopen reaction tube for 5 min at RT. The samples should not be dried in aspeed vacuum! Dissolve pellet in 8 μl Tris-buffer (pH 8.5) and place onice.

Example 1F Amplification by in Vitro-Transcription

In vitro transcription: Template DNA 8 μl ATP/CTP/GTP/UTP (75 mM each) 2μl 10 × buffer 2 μl T7 RNA polymerase 2 μl

Thaw all components and mix them at room temperature, and not on ice,because the spermidine component of the reaction buffer would induceprecipitation of the template. Use 0.5 ml or 0.2 ml RNase-free PCR tubesfor this step.

Incubate the transcription reaction overnight at 37° C. either in athermocycler with heated lid (at 37° C.) or in a hybridisation oven.Load 1-2 μl of the reaction mix onto a 1.5% native agarose gel. Add 1 μlDNase to the remaining reaction and incubate for further 15 min at 37°C. To purify the RNA, use the RNeasy kit from Qiagen according to themanufacturer's protocol for RNA-clean-up. At the end of the clean-upprocedure, elute the RNA by using 2×50 μl DEPC-water and perform anethanol precipitation as described above in step 6. Dissolve RNA pelletin 5 μl DEPC water.

The RNA is now ready for labelling and use in a microarray hybridisationor for further amplification by a second amplification round.

Example 2 Second Amplification Round (See, e.g., FIG. 1 c) Example 2AReverse Transcription of Amplified RNA with the Box Primer

First strand-DNA-synthesis RNA of the fist amplification round 4 μl Boxprimer (5 pmol/μl) 2 μl dNTP-Mix (10 mM) 1 μl DEPC-H₂O 2 μl

Incubate 4 min at 65° C. in a thermocycler with a heated lid, then placeimmediately on ice. Mastermix for synthesis of the first strand cDNA 5 ×RT-buffer 4 μl RNase-Inhibitor (20 U/μl) 1 μl DEPC-H₂O 5 μl

Pipette components for the mastermix on ice and add to the tubecontaining the reverse transcription mix. Place samples in athermocycler (preheated to 48° C.)

Incubate as follows:

-   48° C./1 minute-   cool to 45° C.-   add 1 μl reverse transcriptase (5 U/μl) to each sample-   45° C./30 minutes-   70° C./15 minutes (enzyme inactivation)

Place samples on ice.

Example 2B RNA Removal

Removal of RNA from the reaction First strand cDNA mix 20 μl RNase-Mix(RNase H/RNase I; each at 5 U/μl)  1 μl

Incubate for 20 min at 37° C., hereafter place samples on ice. RNase Awas not used for RNA elimination, because RNase A is not readilyinactivated. RNase I on the other hand, the enzyme used in thisinvention, can be inactivated easily and completely by incubation at 70°C. for 15 min.

Example 2C Double-Stranded Template DNA with T7-(dT)₁₈V Primer

Template DNA from first.strand cDNA with T7-(dT)₁₈V primer Firststrand-cDNA 21 μl  dNTP-mix (10 mM) 1 μl T7-(dT)₁₈V primer (10 pmol/μl)2 μl 10 × polymerase buffer 6 μl H₂O 20 μl 

Incubation:

-   96° C./1 minute-   42° C./1 minute-   add 1 μl reverse transcriptase (5 U/μl) to each sample-   42° C./30 minutes

Generation of blunt ends in dsDNA

-   Cool samples to 37° C.-   add 1 μl T4 DNA polymerase (10 U/μl) to each sample-   37° C./3 minutes-   96° C./15 minutes

Place samples on ice

Example 2D Purification of cDNA with High-Pure PCR Purification Kit(Roche)

cDNA purification Reaction mix  50 μl Binding-buffer 250 μl Carrier(cot-1-DNA, 100 ng/μl)  3 μl

Transfer mix onto provided columns, spin in a tabletop centrifuge atmaximal rpm for 1 min. Discard the flow-through. Add 500 μl washingbuffer to the column and spin as above, discard flow-through and repeatthe wash step with 200 μl washing buffer. Transfer columns onto a new1.5 ml reaction tube add 50 μl elution buffer, incubate for 1 min at RTand centrifuge as described above. Repeat the elution step once, againusing 50 μl buffer.

Example 2E Ethanol Precipitation of Purified cDNA

Do not vortex the Pellet Paint™-carrier stock solution and store in thedark. Keep at −20° C. for long term storage, smaller aliquots can bestored for approximately 1 month at 4° C. Ethanol precipitation Eluate100 μl Carrier (Pellet Paint ™)  2 μl Sodium acetate  10 μl Ethanol;absolute 220 μl

Mix thoroughly (do not vortex) and pellet cDNA by centrifugation atmaximal rpm for 10 min at RT.

Discard supernatant; wash pellet once with 200 μl of 70% ethanol.Centrifuge for 1 min as described above. Remove supernatant completelyusing a pipette. Dry pellet by incubation of the open reaction tube for5 min at RT. The samples should not be dried in a speed vacuum! Dissolvepellet in 8 μl Tris-buffer (pH 8.5) and place on ice.

Example 2F Second Amplification by In Vitro-Transcription

In vitro transcription: Template DNA 8 μl ATP/CTP/GTP/UTP (75 mM each) 2μl 10x buffer 2 μl T7 RNA polymerase 2 μl

Thaw all components and mix them at RT, and not on ice, because thespermidine component of the reaction buffer would induce precipitationof the template. Use 0.5 ml or 0.2 ml RNase-free PCR tubes for thisstep.

Incubate the transcription reaction overnight at 37° C. either in athermocycler with heated lid (at 37° C.) or in a hybridisation oven.Load 1-2 μl of the reaction mix onto a 1.5% native agarose gel. Add 1 μlDNase to the remaining reaction and incubate for further 15 min at 37°C. To purify the RNA, use the RNeasy kit from Qiagen according to themanufacturer's protocol for RNA-clean-up. At the end of the clean-upprocedure, elute the RNA by using 2×50 μl DEPC-water and perform anethanol precipitation as described above in step 6. Dissolve RNA pelletin 5 μl DEPC water.

The RNA is now ready for labelling and use in a microarray hybridisationor for further amplification by a third amplification round (a thirdamplification round is exactly performed as described in Examples2A-2F).

1-37. (canceled)
 38. A method for the amplification of ribonucleic acidscomprising the following steps: a) a single stranded DNA is producedfrom an RNA by means of reverse transcription, using a single-strandedprimer having a defined sequence, an RNA-dependent DNA polymerase anddeoxyribonucleoside triphosphates; b) the template RNA is removed; c) aDNA duplex is produced by means of a single-stranded primer, a DNApolymerase and deoxyribonucleoside triphosphates, wherein the asingle-stranded primer comprises a box sequence and a sequence of 3 to 9oligonucleotides of random sequence, wherein the Box sequence is presentin the 5′ region of the single stranded primer and comprises a sequencewith a length of 10 to 25 nucleotides having a low homology to sequencesexpressed by organisms from which the RNA to be amplified was obtained;d) the duplex is separated into single-stranded DNAs; e) DNA duplexesare produced from one of the single-stranded DNAs obtained in step (d)by means of a single-stranded primer comprising a promoter sequence atits 5′end and the same defined sequence as the primer used in step (a)at its 3′end, a DNA polymerase and deoxyribonucleoside triphosphates; f)a plurality of single stranded RNAs is produced, both ends of whichcomprise defined sequences, by means of an RNA polymerase andribonucleoside triphosphates.
 39. The method according to claim 38,wherein the single-stranded RNA obtained have the inverse senseorientation (antisense sequence) in relation to the RNA startingmaterial.
 40. The method according to claim 38, characterised in thatthe single-stranded primer used in step (a) contains anoligo-dT-sequence.
 41. The method according to claim 38, characterisedin that a 5′-(dT)₁₈V-primer is used in step (a) for reversetranscription, with V being any deoxyribonucleotide-monomer apart fromdT.
 42. The method according to claim 38, characterised in that in step(b) the RNA is hydrolysed by means of RNase.
 43. The method according toclaim 38, characterised in that in step (b) the RNA is removed by meansof RNase I and/or RNase H.
 44. The method according to claim 38,characterised in that in step (c) a single-stranded primer is used withthe following sequence: GCA TCA TAC AAG CTT GGT ACC NNN NNN TCT (30 nt).45. The method according to claim 38, characterised in that a reversetranscriptase is used as DNA polymerase.
 46. The method according toclaim 38, characterised in that dATP, dCTP, dGTP and dTTP are used asdeoxyribonucleotide-monomers.
 47. The method according to claim 38,characterised in that in step (d) DNA double strands are separated insingle strands by means of heat.
 48. The method according to claim 38,characterised in that in step (e) a single-stranded primer is used,which comprises the sequence of either the T7, T3 or SP6 RNA polymerase.49. The method according to claim 38, characterised in that in step (e)a single-stranded primer is used, containing not only a promotersequence but also an oligo(dT)-sequence of at least 8 nucleotides. 50.The method according to claim 38, characterised in that in step (e) thesingle-stranded primer has the following sequence: ACT AAT ACg ACT CACTAT A g⁺¹ g (dT)₁₈V (40 nt).
 51. The method according to claim 38,characterised in that in step (f) T7 RNA polymerase is used as RNApolymerase
 52. The method according to claim 38, characterised in thatATP, CTP, GTP and UTP are used as ribonucleotide-monomers.
 53. Themethod according to claim 38, characterised in that the amplificationfactor of the starting RNA sequence is at least 500, preferably morethan
 1000. 54. The method according to claim 38, characterised in thatthe method comprises after step (f) the following steps for furtheramplification of ribonucleic acids: g) using the in step (f) generatedsingle-stranded RNAs as template, single-stranded DNA is synthesisedusing reverse transcriptase, a single-stranded primer, containing theBox sequence, an RNA-dependant DNA polymerase and deoxyribonucleosidetriphosphates; h) the RNA is removed; i) using the in (h) generatedsingle-stranded DNA as template, double-stranded DNA is synthesisedusing a single-stranded primer, comprising a promoter sequence in its 5′region and the same defined sequence as the primer used in step (a), inits 3′ region, a DNA polymerase and deoxyribonucleoside triphosphates;j) a multitude of single-stranded RNAs is synthesized using a RNApolymerase and ribonucleoside triphosphates.
 55. The method according toclaim 54, characterised in that in step (i) the single stranded primeris identical with the single-stranded primer used in step (e).
 56. Themethod according to claim 54, characterised in that in step (h) the RNAis hydrolysed by means of RNase.
 57. The method according to claim 54,characterised in that all single-stranded RNAs produced in step (O) haveinverse orientation.
 58. A kit for ribonucleic acid amplificationaccording to the method of claim 38, comprising the followingcomponents: a) at least at least one single-stranded primer comprising apromoter sequence; b) at least one single-stranded primer comprising abox sequence; c) an RNA-dependent DNA polymerase; d) deoxyribonucleosidetriphosphates; e) a DNA-dependent DNA polymerase; f) an RNA polymerase;and g) ribonucleoside triphosphates.
 59. The kit according to claim 58,characterised in that the kit comprises three different single-strandedprimers.
 60. The kit according to claim 58, characterised in that thesingle-stranded primer comprising the promoter sequence, also comprisesan oligo-dT-sequence.
 61. The kit according to claim 58, characterisedin that a single-stranded primer comprises a 5′-(dT)₁₈V-primer sequencefor reverse transcription, with V being any deoxyribonucleotide-monomerapart from dT.
 62. The kit according to claim 58, characterised in thatin addition, the kit comprises RNase I and/or RNase H.
 63. The kitaccording to claim 58, characterised in that the kit comprises asingle-stranded primer with a T7, T3 or SP6 RNA polymerase promotersequence.
 64. The kit according to claim 58, characterised in that asingle-stranded primer is used with the following sequence: ACT AAT ACgACT CAC TAT A g⁺¹ g (dT)₁₈V (40 nt).
 65. The kit according to claim 58,characterised in that it comprises a reverse transcriptase as DNApolymerase.
 66. The kit according to claim 58, characterised in that itcomprises the T7 RNA polymerase.
 67. The kit according to claim 58,characterised in that it comprises a composition for labelling of DNAwith a detectable moiety.
 68. The kit according to claim 58,characterised in that the kit includes a DNA-microarray.
 69. A methodfor nucleic acid analysis that involves production of ribonucleic acids,amplification with the method according to claim 38, and analysis bymeans of microarrays.
 70. The method according to claim 69,characterised in that the ribonucleic acids is isolated from abiological sample.
 71. The method according to claim 69, characterisedin that ribonucleic acids are amplified, converted to cDNA by means ofreverse transcription, and the cDNAs are analysed by means ofmicoarrays.
 72. The method according to claim 69, characterised in thatthe amount and/or sequence of the cDNA are analysed.